Several factors contribute to the pathogenesis of diabetic nephropathy (DN), including hyperglycemia, advanced glycosylation end products (AGEs), and the rennin angiotensin system. These factors can lead to glomerular expansion and extracellular matrix (ECM) accumulation - the hallmark of DN. However, the specific molecular and signaling mechanisms for these processes are not clear. High glucose (HG) and Angiotensin (AngII) can activate the lipoxygenase (LO) pathway of arachidonate metabolism in vascular smooth muscle cells. 12-LO products have growth-promoting products and can activate protein kinase C (PKC) as well as mitogen activated protein kinases (MAPKs). The major hypothesis is that the 12-LO pathway activated by high glucose (HG) and AngII plays a key role in the pathogenesis of DN by regulating the activation of PKC and downstream effector MAPKs in mesangial cells. These events may alter ECM and growth factor synthesis (TGFB1) in key sites such as mesangial cells (MC) which play an important role in DN. 12-LO will be studied since it is the major LO pathway in MC. The specific Aims are 1) To examine the regulation of 12-LO activation and expression in MC by HG (25 mM), AGEs, and AngII. 2) To determine the functional significance of 12-LO activation in rat MC. We will test whether 12-LO products directly increase ECM protein fibronection and TGFB1. We will then test whether the12-LO pathway mediates ECM induced by HG and Ang II by examining the effects of a specific molecular LO inhibitor, namely, a 12-LO ribozyme, as well as examine MC from 12-LO knock out mice. Conversely, we will determine whether over-expressionof 12-LO can increase ECM production in MC by testing adenoviral vector-mediated over-expression of mouse 12-LO in rat MC, ad by testing MC from a new 12-LO transgenic mouse model. 3) To evaluate the signal transduction mechanisms by which 12-LO products mediate pathologic effects in MC. In particular, the role of p38MAPK will be examined. 4) To examine the in vivo relevance of 12-LO activation in the development of DN. We will determine whether 12-LO, p38MAPK and ECM levels are increased in glomeruli from diabetic rats. We will then test the hypothesis that 12-LO knockout mice will develop milder or less frequent diabetic renal complications relative to genetically matched controls, and conversely, 12-LO transgenic mice will develop more severe diabetic renal complications relative to non-transgenic littermates. Preliminary data showing trophic effects of 12-LO products in vitro in MC, as well as increased expression of 12-LO and p38MAPK family members in vivo in glomeruli from diabetic rats provide strong support for the hypothesis. These integrated in vitro and in vivo studies will provide new information on the mechanisms of glomerular expansion in DN, and thus lead to the development of novel therapies to combat this major complication of diabetes.